Cells expressing eGFP-tagged Rab5 (wild-type or the GDP-Rab5 mutant) and the DsRed-tagged α-adrenergic receptors were employed and the roles of GRK2 were studied utilizing paroxetine and the dominant-negative mutant of GRK2 (DN-GRK2). The following parameters were studied: a) FRET (as an index of α-adrenergic receptor-Rab5 interaction): b) intracellular accumulation of DsRed fluorescence (receptor internalization); c) α-adrenergic receptor phosphorylation, and d) noradrenaline-induced increase in intracellular calcium concentration. Noradrenaline increased α-adrenergic receptor-Rab5 interaction, which was blocked by paroxetine and by expression of the dominant-negative GRK2 mutant. Similarly, paroxetine and expression of the DN-GRK2 or the GDP-Rab5 mutants markedly decreased receptor internalization, α-adrenergic receptor phosphorylation, and attenuated the ability of the adrenergic agonist to induce homologous desensitization (calcium signaling). The S406, 410,412A α-adrenergic receptor mutant did not reproduce the actions of GRK2 inhibition. The data indicate that GRK2 and Rab5 play key roles in α-adrenergic receptor phosphorylation, internalization, and desensitization. The possibility that Rab5 might form part of a signaling complex is suggested, as well as that GDP-Rab5 might interfere with the ability of GRK2 to catalyze α-adrenergic receptor phosphorylation.